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. 1995 Aug 15;92(17):7739–7743. doi: 10.1073/pnas.92.17.7739

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

B A Bunnell 1, L M Muul 1, R E Donahue 1, R M Blaese 1, R A Morgan 1
PMCID: PMC41221  PMID: 7644487

Abstract

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson W. French, Blaese R. Michael, Culver Kenneth. The ADA human gene therapy clinical protocol: Points to Consider response with clinical protocol, July 6, 1990. Hum Gene Ther. 1990 Fall;1(3):331–362. doi: 10.1089/hum.1990.1.3-331. [DOI] [PubMed] [Google Scholar]
  2. Culver K. W., Morgan R. A., Osborne W. R., Lee R. T., Lenschow D., Able C., Cornetta K., Anderson W. F., Blaese R. M. In vivo expression and survival of gene-modified T lymphocytes in rhesus monkeys. Hum Gene Ther. 1990 Winter;1(4):399–410. doi: 10.1089/hum.1990.1.4-399. [DOI] [PubMed] [Google Scholar]
  3. Culver K., Cornetta K., Morgan R., Morecki S., Aebersold P., Kasid A., Lotze M., Rosenberg S. A., Anderson W. F., Blaese R. M. Lymphocytes as cellular vehicles for gene therapy in mouse and man. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3155–3159. doi: 10.1073/pnas.88.8.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Goodman S., Xiao X., Donahue R. E., Moulton A., Miller J., Walsh C., Young N. S., Samulski R. J., Nienhuis A. W. Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells. Blood. 1994 Sep 1;84(5):1492–1500. [PubMed] [Google Scholar]
  5. Hock R. A., Miller A. D., Osborne W. R. Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines. Blood. 1989 Aug 1;74(2):876–881. [PubMed] [Google Scholar]
  6. Johann S. V., Gibbons J. J., O'Hara B. GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus. J Virol. 1992 Mar;66(3):1635–1640. doi: 10.1128/jvi.66.3.1635-1640.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kasid A., Morecki S., Aebersold P., Cornetta K., Culver K., Freeman S., Director E., Lotze M. T., Blaese R. M., Anderson W. F. Human gene transfer: characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man. Proc Natl Acad Sci U S A. 1990 Jan;87(1):473–477. doi: 10.1073/pnas.87.1.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kavanaugh M. P., Miller D. G., Zhang W., Law W., Kozak S. L., Kabat D., Miller A. D. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7071–7075. doi: 10.1073/pnas.91.15.7071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kotani H., Newton P. B., 3rd, Zhang S., Chiang Y. L., Otto E., Weaver L., Blaese R. M., Anderson W. F., McGarrity G. J. Improved methods of retroviral vector transduction and production for gene therapy. Hum Gene Ther. 1994 Jan;5(1):19–28. doi: 10.1089/hum.1994.5.1-19. [DOI] [PubMed] [Google Scholar]
  10. Mavilio F., Ferrari G., Rossini S., Nobili N., Bonini C., Casorati G., Traversari C., Bordignon C. Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer. Blood. 1994 Apr 1;83(7):1988–1997. [PubMed] [Google Scholar]
  11. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Miller A. D., Garcia J. V., von Suhr N., Lynch C. M., Wilson C., Eiden M. V. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol. 1991 May;65(5):2220–2224. doi: 10.1128/jvi.65.5.2220-2224.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  14. Morgan R. A., Anderson W. F. Human gene therapy. Annu Rev Biochem. 1993;62:191–217. doi: 10.1146/annurev.bi.62.070193.001203. [DOI] [PubMed] [Google Scholar]
  15. Morgan R. A., Baler-Bitterlich G., Ragheb J. A., Wong-Staal F., Gallo R. C., Anderson W. F. Further evaluation of soluble CD4 as an anti-HIV type 1 gene therapy: demonstration of protection of primary human peripheral blood lymphocytes from infection by HIV type 1. AIDS Res Hum Retroviruses. 1994 Nov;10(11):1507–1515. doi: 10.1089/aid.1994.10.1507. [DOI] [PubMed] [Google Scholar]
  16. Morgan R. A., Cornetta K., Anderson W. F. Applications of the polymerase chain reaction in retroviral-mediated gene transfer and the analysis of gene-marked human TIL cells. Hum Gene Ther. 1990 Summer;1(2):135–149. doi: 10.1089/hum.1990.1.2-135. [DOI] [PubMed] [Google Scholar]
  17. Woffendin C., Yang Z. Y., Udaykumar, Xu L., Yang N. S., Sheehy M. J., Nabel G. J. Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11581–11585. doi: 10.1073/pnas.91.24.11581. [DOI] [PMC free article] [PubMed] [Google Scholar]

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